Cellulose synthesis in maize: isolation and expression analysis of the cellulose synthase (CesA) gene family

Stalk lodging in maize results in significant yield losses. We have determined that cellulose per unit length of the stalk is the primary determinant of internodal strength. An increase in cellulose concentration in the wall might allow simultaneous improvements in stalk strength and harvest index. Cellulose formation in plants can be perturbed by mutations in the genes involved in cellulose synthesis, post-synthetic cellulose alteration or deposition, N-glycosylation, and some other genes with as yet unknown functions. We have isolated 12 members of the cellulose synthase (CesA) gene family from maize. The genes involved in primary wall formation appear to have duplicated relatively independently in dicots and monocots. The deduced amino acid sequences of three of the maize genes, ZmCesA10–12, cluster with the Arabidopsis CesA sequences that have been shown to be involved in secondary wall formation. Based on their expression patterns across multiple tissues, these three genes appear to be coordinately expressed. The remaining genes show overlapping expression to varying degrees with ZmCesA1, 7, and 8 forming one group, ZmCesA3 and 5 a second group, and ZmCesA2 and 6 exhibiting independent expression of any other gene. This suggests that the varying levels of coexpression may just be incidental except in the case of ZmCesA10–12, which may interact with each other to form a functional enzyme complex. Isolation of the expressed CesA genes from maize and their association with primary or secondary wall formation has made it possible to test their respective roles in cellulose synthesis through mutational genetics or transgenic approaches. This information would be useful in improving stalk strength.     

Identification and expression analysis of genes encoding putative cellulose synthases (CesA) in the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.)

Cellulose is synthesized in plant cell walls by large membrane-bound protein complexes proposed to contain several copies of the catalytic subunit of the cellulose synthase, CesA. Here we report identification of 10 distinct CesA genes within a database of 100,000 ESTs of the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.). Expression analyses in normal wood undergoing xylogenesis and in tension wood indicate xylem specific expression of four putative CesA isoenzymes, PttCesA1, PttCesA3-1, PttCesA3-2 and PttCesA9. Both the protein sequences and the expression profiles of PttCesA3-1 and PttCesA3-2 are very similar, and they may thus represent redundant copies of an enzyme with essentially the same function. Further, one of the generally more constitutively expressed CesA genes, PttCesA2, seems to be activated on the opposite side of a tension wood induced stem, while PttCesA6 appears to be more specific for leaf tissues. The rest of the hybrid aspen CesA genes were found to be relatively evenly expressed over the poplar tissues hereby studied.     

Cellulose synthase (CesA) genes in algae and seedless plants

Microfibril structure is determined largely by the organization of arrays of integral plasma membrane protein particles known as “terminal complexes”, which include cellulose synthase catalytic subunits encoded by CesA genes. Although the CesA genes of plants and bacteria share conserved regions, variations in terminal complex and microfibril structure presumably result from sequence differences. Thus, the CesA domains that influence terminal complex assembly may be revealed by examining the differences between CesA genes from green algae in which terminal complex structure ranges from rosettes (plant-like) to linear (bacteria-like). This report describes a second CesA gene that has been cloned from Mesotaenium caldariorum, a unicellular green alga from the order Zygnematales, which have rosette terminal complexes. Both McCesA1 and McCesA2 are similar to seed plant CesAs in domain structure and intron position. Seed plants have multiple CesAs and CesA-like (Csl) genes, some of which appear to be expressed specifically during cell expansion, secondary cell wall deposition in vascular tissue, or tip growth. Diversification of the CesA and Csl gene families can be explored by comparing these genes in mosses, which lack vascular tissue with secondary cell walls, and early divergent vascular plants such as ferns. Degenerate primers were used to amplify and clone five unique CesA and Csl fragments from genomic DNA isolated from Physcomitrella patens. Probes derived from the cloned fragments were used to isolate several clones from a Physcomitrella genomic library. One Csl fragment was amplified from genomic DNA isolated from the fern Ceratopteris richardii. Phylogenetic analysis supports the presence of CslD genes in both mosses and ferns, but does not support the presence of secondary cell wall specific CesA orthologs in mosses.     

Updating Insights into the Catalytic Domain Properties of Plant Cellulose synthase (CesA) and Cellulose synthase-like (Csl) Proteins

The wall is the last frontier of a plant cell involved in modulating growth, development and defense against biotic stresses. Cellulose and additional polysaccharides of plant cell walls are the most abundant biopolymers on earth, having increased in economic value and thereby attracted significant interest in biotechnology. Cellulose biosynthesis constitutes a highly complicated process relying on the formation of cellulose synthase complexes. Cellulose synthase (CesA) and Cellulose synthase-like (Csl) genes encode enzymes that synthesize cellulose and most hemicellulosic polysaccharides. Arabidopsis and rice are invaluable genetic models and reliable representatives of land plants to comprehend cell wall synthesis. During the past two decades, enormous research progress has been made to understand the mechanisms of cellulose synthesis and construction of the plant cell wall. A plethora of cesa and csl mutants have been characterized, providing functional insights into individual protein isoforms. Recent structural studies have uncovered the mode of CesA assembly and the dynamics of cellulose production. Genetics and structural biology have generated new knowledge and have accelerated the pace of discovery in this field, ultimately opening perspectives towards cellulose synthesis manipulation. This review provides an overview of the major breakthroughs gathering previous and recent genetic and structural advancements, focusing on the function of CesA and Csl catalytic domain in plants.     

How the geometrical model for plant cell wall formation enables the production of a random texture

Cellulose synthase (CESA) molecules are the building blocks and catalytic centers of the CESA complex. The study of mutants in Arabidopsis has led to insight into the structure of these nanomachines. Inside the plasma membrane, the CESA molecules are arranged in complexes, which, apart from the CESA molecules proper, contain other, mostly unidentified, proteins. We developed a theory in which CESA density, together with distance between cellulose microfibrils (CMFs) being deposited and cell geometry, determines wall texture. We have shown earlier how this theory is able to explain the production of axial, helical, helicoid and crossed-polylamellate textures. In the present article we extend this theory to random wall textures.     

Prediction of the structures of the plant-specific regions of vascular plant cellulose synthases and correlated functional analysis

Seed plants express cellulose synthase (CESA) protein isoforms with non-redundant functions, but how the isoforms function differently is unknown. Compared to bacterial cellulose synthases, CESAs have two insertions in the large cytosolic loop: the relatively well-conserved Plant Conserved Region (P-CR) and a Class Specific Region (CSR) that varies between CESAs. Absent any atomic structure of a plant CESA, we used ab initio protein structure prediction and molecular modeling to explore how these plant-specific regions may modulate CESA function. We modeled P-CR and CSR peptides from Arabidopsis thaliana CESAs representing the six clades of seed plant CESAs. As expected, the predicted wild type P-CR structures were similar. Modeling of the mutant P-CR of Atcesa8 ^R362K (fra6) suggested that changes in local structural stability and surface electrostatics may cause the mutant phenotype. Among CSRs within CESAs required for primary wall cellulose synthesis, the amino sequence and the modeled arrangement of helices was most similar in AtCESA1 and AtCESA3. Genetic complementation of known Arabidopsis mutants showed that the CSRs of AtCESA1 and AtCESA3 can function interchangeably in vivo. Analysis of protein surface electrostatics led to ideas about how the surface charges on CSRs may mediate protein–protein interactions. Refined modeling of the P-CR and CSR regions of GhCESA1 from cotton modified their tertiary structures, spatial relationships to the catalytic domain, and preliminary predictions about CESA oligomer formation. Cumulatively, the results provide structural clues about the function of plant-specific regions of CESA.     

Global expression analysis of CESA and CSL genes in Arabidopsis

We have used Affymetrix gene chips to measure the expression of 10 CESA and 29 CSL genes of Arabidopsis in different developmental stages or organs. These measurements reveal that many of the genes exhibit different levels of expression in the various organs. While several CESA genes are highly expressed in all the tissues examined, very few CSL genes approach such high levels of expression. This suggests that the CSL genes either encode enzymes for the synthesis of minor components of cell walls or are expressed only in specific cell types. The expression data also highlights the potential importance of the CESA genes for primary and secondary cell wall formation during different developmental stages and in the different organs examined.     

Cellulose synthesis in the Arabidopsis secondary cell wall

The identification of genes responsible for cellulose synthesis has led to a significant advance in our understanding of the synthesis of this important polymer. The identification of these genes has arisen from the identification of cellulose deficient mutants. The irregular xylem (irx) mutants of Arabidopsis are caused by a severe reduction in cellulose synthesis in the secondary cell wall. Three irx mutants deficient in secondary cell wall cellulose are the result of mutations in three different members of the cellulose synthase catalytic subunit (CesA) gene family. The three proteins encoded by these genes all associate within the same membrane bound complex, and the presence of all three, but not their activity, is required for correct assembly and targeting of this complex. The knowledge that these three proteins associate provides a good opportunity to purify the cellulose synthase complex, and recent results working towards this goal are discussed.     

Isolation and characterization of the cellulose synthase genes <Emphasis Type="Italic">PpCesA6</Emphasis> and <Emphasis Type="Italic">PpCesA7</Emphasis> in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Analysis of cellulose biosynthesis using molecular approaches has been successful in identifying genes in many cellulose-producing organisms, yet the mechanism of cellulose biosynthesis still remains to be understood. We are interested in developing the moss Physcomitrella patens as a useful system for the study of cellulose biosynthesis. This moss affords a number of advantages including a haploid dominated gametophyte and a very high efficiency of homologous recombination in its nuclear DNA for constructing gene knockouts. In addition, P. patens has only a primary cell wall unlike Arabidopsis thaliana, which has both a primary and a secondary cell wall. We identified two full-length cellulose synthase (CesA) genes of P. patens, PpCesA6 and PpCesA7 from an EST database and have analyzed the genomic sequences. PpCesA6 and PpCesA7 show high similarity to each other, both at the cDNA and genomic DNA levels. Single and double knockouts of PpCesA6 and PpCesA7 were generated and screened for phenotypic changes. While the PpCesA6 and PpCesA7 single knockouts did not show any obvious phenotypic differences from the wild-type, the double knockout had significantly reduced stem length. These results suggest that PpCesA6 and PpCesA7 probably have a very similar role in cellulose biosynthesis and their functions may be redundant. Additionally, their roles may overlap with the other P. patens CesAs as observed for CesAs involved in primary cell wall biosynthesis in A. thaliana.     

Selective delignification of wood by white-rot fungi

The white-rot fungi,Cerrena unicolor, Ganoderma applanatum, G. tsugae,Ischnoderma resinosum, andPerenniporia medullapanis, caused two distinct types of decay. Large areas of decayed wood were selectively delignified and a typical white-rot causing a simultaneous removal of all cell wall components was present. Preferential lignin degradation was intermittently dispersed throughout the decayed wood. Scanning and transmission electron microscopy were used to identify the micromorphological and ultrastructural changes that occurred in the cells during degradation. In delignified areas the compound middle lamella was extensively degraded without substantial alteration of the secondary wall. The S₂ layer of the secondary wall was least affected. The loss of middle lamellae resulted in extensive defibration of the cells. Sulfuric acid lignin determinations indicated that 95–98% of the lignin was removed. Wood sugar analyses using high pressure liquid chromatography demonstrated that hemicelluloses were removed in preference to cellulose when lignin was degraded. The results suggest that a highly diffusible ligninolytic system was responsible for the selective degradation of the wood. In simultaneously white-rotted wood, all cell wall layers were progressively removed from the cell lumen toward the middle lamella, causing erosion troughs or holes to form. Large voids filled with fungal mycelia resulted from a coalition of degraded areas. Chemical analyses of white-rotted wood indicated lignin, cellulose, and hemicellulose were removed in approximately the same amounts. Degradation was confined to areas around fungal hyphae.     

Xylem-specific and tension stress-responsive expression of cellulose synthase genes from aspen trees

Genetic improvement of cellulose biosynthesis in woody trees is one of the major goals of tree biotechnology research. Yet, progress in this field has been slow owing to (1) unavailability of key genes from tree genomes, (2) the inability to isolate active and intact cellulose synthase complexes and, (3) the limited understanding of the mechanistic processes involved in the wood cellulose development. Here I report on the recent advances in molecular genetics of cellulose synthases (CesA) from aspen trees. Two different types of cellulose synthases appear to be involved in cellulose deposition in primary and secondary walls in aspen xylem. The three distinct secondary CesAs from aspen—PtrCesA1, PtrCesA2, and PtrCesA3—appear to be aspen homologs of Arabidopsis secondary CesAs AtCesA8, AtCesA7, and AtCesA4, respectively, based on their high identity/similarity (>80%). These aspen CesA proteins share the transmembrane domain (TMD) structure that is typical of all known “true” CesA proteins: two TMDs toward the N-terminal and six TMDs toward the C-terminal. The putative catalytic domain is present between TMDs 2 and 3. All signature motifs of processive glycosyltransferases are also present in this catalytic domain. In a phylogenetic tree based on various predicted CesA proteins from Arabidopsis and aspen, aspen CesAs fall into families similar to those seen with Arabidopsis CesAs, suggesting their functional similarity. The coordinate expression of three aspen secondary CesAs in xylem and phloem fibers, along with their simultaneous tension stress-responsive upregulation, suggests that these three CesAs may play a pivotal role in biosynthesis of better-quality cellulose in secondary cell walls of plants. These results are likely to have a direct impact on genetic manipulation of trees in the future.     

2D time-domain nuclear magnetic resonance (2D TD-NMR) characterization of cell wall water of Fagus sylvatica and Pinus taeda L.

Cellulose - Water dominates the wood cell wall in the hygroscopic range. The state and amount of cell wall water influence the physical and mechanical properties of wood. 2D time-domain nuclear...     

Genetic and expression alterations in association with the sarcomatous change of cholangiocarcinoma cells

Cholangiocarcinoma (CC) is an intrahepatic bile duct carcinoma with a high mortality rate and a poor prognosis. Sarcomatous change/epithelial mesenchymal transition (EMT) of CC frequently leads to aggressive intrahepatic spread and metastasis. The aim of this study was to identify the genetic alterations and gene expression pattern that might be associated with the sarcomatous change in CC. Previously, we established 4 human CC cell lines (SCK, JCK1, Cho-CK, and Choi-CK). In the present study, we characterized a typical sarcomatoid phenotype of SCK, and classified the other cell lines according to tumor cell differentiation (a poorly differentiated JCK, a moderately differentiated Cho-CK, and a well differentiated Choi-CK cells), both morphologically and immunocytologically. We further analyzed the genetic alterations of two tumor suppressor genes (p53 and FHIT) and the expression of Fas/FasL gene, well known CC-related receptor and its ligand, in these four CC cell lines. The deletion mutation of p53 was found in the sarcomatoid SCK cells. These cells expressed much less Fas/FasL mRNAs than did the other ordinary CC cells. We further characterize the gene expression pattern that is involved in the sarcomatous progression of CC, using cDNA microarrays that contained 18,688 genes. Comparison of the expression patterns between the sarcomatoid SCK cells and the differentiated Choi-CK cells enabled us to identify 260 genes and 247 genes that were significantly over-expressed and under-expressed, respectively. Northern blotting of the 14 randomly selected genes verified the microarray data, including the differential expressions of the LGALS1, TGFBI, CES1, LDHB, UCHL1, ASPH, VDAC1, VIL2, CCND2, S100P, CALB1, MAL2, GPX1, and ANXA8 mRNAs. Immunohistochemistry also revealed in part the differential expressions of these gene proteins. These results suggest that those genetic and gene expression alterations may be relevant to the sarcomatous change/EMT in CC cells.     

On the organization of hemicelluloses in the wood cell wall

Cellulose - The structural arrangement of the polymers in the cell wall of wood has still not been fully established. This relates specifically to the role of the two hemicelluloses, glucomannan...     

Inhibition of mouse brown adipocyte differentiation by second-generation antipsychotics

Brown adipose tissue is specialized to burn lipids for thermogenesis and energy expenditure. Second-generation antipsychotics (SGA) are the most commonly used drugs for schizophrenia with several advantages over first-line drugs, however, it can cause clinically-significant weight gain. To reveal the involvement of brown adipocytes in SGA-induced weight gain, we compared the effect of clozapine, quetiapine, and ziprasidone, SGA with different propensities to induce weight gain, on the differentiation and the expression of brown fat-specific markers, lipogenic genes and adipokines in a mouse brown preadipocyte cell line. On Oil Red-O staining, the differentiation was inhibited almost completely by clozapine (40 µM) and partially by quetiapine (30 µM). Clozapine significantly down-regulated the brown adipogenesis markers PRDM16, C/EBPβ, PPARγ2, UCP-1, PGC-1α, and Cidea in dose- and time-dependent manners, whereas quetiapine suppressed PRDM16, PPARγ2, and UCP-1 much weakly than clozapine. Clozapine also significantly inhibited the mRNA expressions of lipogenic genes ACC, SCD1, GLUT4, aP2, and CD36 as well as adipokines such as resistin, leptin, and adiponectin. In contrast, quetiapine suppressed only resistin and leptin but not those of lipogenic genes and adiponectin. Ziprasidone (10 µM) did not alter the differentiation as well as the gene expression patterns. Our results suggest for the first time that the inhibition of brown adipogenesis may be a possible mechanism to explain weight gain induced by clozapine and quetiapine.     

Characterization of fossil Sequoioxylon wood using analytical instrumental techniques

In this study, fossil (Sequoioxylon) wood from the Oligocene–Miocene transition in İstanbul, Turkey was examined using non-destructive test methods to evaluate changes in anatomical and chemical structure. Molecular changes in the cell wall structure of the wood were determined using Fourier transform infrared (FTIR) and FT-Raman spectroscopy, along with the comparison to recent wood [Sequoiadendron giganteum (Lindl.)]. We found that the cell wall carbohydrates of the fossil wood were significantly more degraded compared with lignin; FT-Raman spectroscopy revealed the degradation in more detail compared with FTIR spectroscopy. FT-Raman spectra also demonstrated that hemicellulose and holocellulose were decreased in the fossil wood. Laser-induced breakdown spectroscopy (LIBS) analysis confirmed that the mass loss was due to the decreased H and O content of the fossil wood sample and was caused by decomposition. Light microscopy also showed that fossil and recent woods have similar anatomic structures, and that the micro-morphological structure of the fossil wood was well-preserved.     

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas in conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.     

Defibration mechanisms of autohydrolyzed Eucalyptus wood chips

The objective of this study was to evaluate the influence of autohydrolysis on mechanical defibration of Eucalyptus wood chips. The autohydrolysis process changed notably the mechanical properties of Eucalyptus chips. The removal of mainly hemicelluloses undoubtedly decreased the overall pulp yield. Hemicellulose losses cannot be solely accounted for the changes in the wood and pulp properties, because the autohydrolysis also caused changes in lignin. When comparing the mechanical pulp fibers of the original wood chips with the fibers resulting from the autohydrolyzed wood material, it was clear that the rupture point shifted from the secondary wall to the middle lamella, confirmed by X-ray photoelectron spectroscopy measurements. This study revealed the mechanical behavior of autohydrolyzed wood chips and can provide useful information for integration of mechanical pulp mills into the biorefinery concept in the future.     

Expression Profiling of Flavonoid Biosynthesis Genes and Secondary Metabolites Accumulation in Populus under Drought Stress

Flavonoids are key secondary metabolites that are biologically active and perform diverse functions in plants such as stress defense against abiotic and biotic stress. In addition to its importance, no comprehensive information has been available about the secondary metabolic response of Populus tree, especially the genes that encode key enzymes involved in flavonoid biosynthesis under drought stress. In this study, the quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the expression of flavonoid biosynthesis genes (PtPAL, Pt4-CL, PtCHS, PtFLS-1, PtF3H, PtDFR, and PtANS) gradually increased in the leaves of hybrid poplar (P. tremula × P. alba), corresponding to the drought stress duration. In addition, the activity and capacity of antioxidants have also increased, which is positively correlated with the increment of phenolic, flavonoid, anthocyanin, and carotenoid compounds under drought stress. As the drought stress prolonged, the level of reactive oxygen species such as hydrogen peroxide (H₂O₂) and singlet oxygen (O₂⁻) too increased. The concentration of phytohormone salicylic acid (SA) also increased significantly in the stressed poplar leaves. Our research concluded that drought stress significantly induced the expression of flavonoid biosynthesis genes in hybrid poplar plants and enhanced the accumulation of phenolic and flavonoid compounds with resilient antioxidant activity.     

Investigation of the multilayered structure and microfibril angle of different types of bamboo cell walls at the micro/nano level using a LC-PolScope imaging system

Bamboo has excellent mechanical properties compared to wood and other plant materials, due to its multilayered structure and polytropic microfibril angle (MFA). The micro/nano scale structure and MFA of fibers, parenchyma cells, and vessels from 4-year-old Moso bamboo (Phyllostachys Heterocycla Var. Pubescens) were investigated by a novel LC-PolScope imaging system and transmission electron microscopy. At the nanoscale, the numbers of layers and accurate MFA for each layer especially thin layers could be obtained quickly using this novel LC-PolScope imaging system. Based on the differences of structure and shape, fibers and parenchyma cells in the vascular bundle were divided into FI, II, III and PI, II cells, respectively. The former class of FI, II, III included 2, 6–8, and 6–8 secondary cell wall layers in turn. The latter class exhibited 9 secondary cell wall layers, with a maximum of 16 layers. To our knowledge, this is the first report of accurate MFA measurement based on the differences of structure and shape for every layer of single fibers, parenchyma cells and vessels in the vascular bundle. For all three cell types, the results also showed that the MFA of sub-layers in secondary walls followed the same changing law: alternating smaller and then bigger MFA. This structural form may be the consequence of natural selection and optimization indicating the long-term mechanical adaptation of bamboo.